Factors Influencing Genetic Transformation and Plant Regeneration of<i>Vitis</i>

Abstract

The effect of callus induction media, culture protocols, embryogenic culture age and cotyledon excision treatment on the production of transgenic embryo and plant lines of <i>Vitis</i> was studied. Embryogenic cultures initiated from leaves or stamens and pistils were transformed with <i>Agrobacterium</i> containing an enhanced green fluorescent protein/neomycin phosphotransferase II (e<i>gfp/nptII</i>) fusion gene. The production of transgenic embryo lines on different culture media was genotype-dependent. <i>Vitis vinifera</i> produced the most transgenic embryo lines (7.5 to 26%) when cultured on DM and X6 medium, whereas <i>Vitis champinii</i> and certain <i>Vitis</i> hybrids produced the best response on NB and X6 medium (5 to 12.5%). Among <i>Vitis vinifera</i> genotypes, Merlot, Superior Seedless, and Thompson Seedless produced transgenic embryo lines irrespective of 4, 8, or 12 month culture ages (11.6 to 36%), whereas Cabernet franc, Cabernet Sauvignon, and Shiraz produced embryo lines only from 4 month cultures (4.1 to 16%). The effect of cotyledon excision of germinated embryos on recovery of transgenic plants varied with variety. The treatment resulted in better plant recovery in <i>Vitis vinifera</i> and <i>Vitis riparia</i> (26.7 to 73.3%), whereas it resulted in a lower plant recovery in <i>Vitis champinii</i> and <i>Vitis</i> hybrids. Transgenic plants were recovered from 19 <i>Vitis</i> genotypes comprised of the species <i>Vitis champinii</i>, <i>Vitis riparia</i>, <i>Vitis rupestris</i>, <i>Vitis vinifera,</i> and <i>Vitis</i> interspecific hybrids. PCR and quantitative real-time PCR confirmed the presence and copy number of the e<i>gfp</i> transgene in selected transgenic plants. Transgenic vines established in the field exhibited normal vegetative and reproductive growth compared to non-transformed vines. Stable GFP (green fluorescence protein) expression patterns in mature plant parts of transgenic plants have been observed for over 4 years indicating no transgene silencing.